Production and characterization of antibody against diacetoxyscirpenol.

Antibodies against diacetoxyscirpenol (DAS) were obtained from rabbits after immunizing them with hemisuccinate or hemiglutarate derivatives of DAS conjugated to bovine serum albumin (BSA). DAS-hemiglutarate-BSA was found to be a much better immunogen than DAS-hemisuccinate-BSA. Competitive radioimmunoassay revealed that the antisera obtained from rabbits after immunization with DAS-hemiglutarate-BSA showed high specificity toward DAS. The concentrations causing 50% displacement of radioactive DAS by unlabeled DAS, 4-monoacetoxyscirpenol (MAS), and 15-MAS were found to be 1.5, 130, and 300 ng per assay, respectively. Thus, the cross-reactivities for 4-MAS and 15-MAS are ca. 87 and 300 times weaker than that of DAS. Practically no cross-reaction (less than 5% displacement) was observed when deoxynivalenol, T-2 toxin, deoxyverrucarol, and scirpentriol were tested at a concentration of 2,000 ng/ml.     

<Emphasis Type="Italic">Streptomyces tritolerans</Emphasis> sp. nov., a novel actinomycete isolated from soil in Karnataka,India

A Gram-positive, nonmotile, moderately halophilic, alkali and thermotolerant strain designated DAS 165(T), was isolated from a dry land soil sample from the Gulbarga region, Karnataka province, India. The isolate produced yellow substrate mycelia and gray aerial mycelia on most tested media. Strain DAS 165(T) showed growth in the presence of 5 to 7% NaCl and at 45 degrees C. The DNA G + C content was 69.7%. 16S rRNA gene sequence analysis together with these characteristics consistently assigned strain DAS 165(T) to the genus Streptomyces. The 16S rRNA gene sequence analysis revealed that strain DAS 165(T) was most closely related to S. tendae ATCC 19812(T) (D 63873) with a sequence similarity of 99.6% (three nucleotide differences out of 1,517). Strain DAS 165(T) formed a distinct clade based on analysis of the almost complete sequence and 120-nucleotide variable gamma region of the 16S rRNA gene. Despite the high sequence similarity, strain DAS 165(T) was phenotypically different from S. tendae ATCC 19812(T). DNA-DNA hybridization between these strains was 47% showing that strain DAS 165(T) is a distinct genomic species. Phenetic and genetic results support the classification of strain DAS 165(T) as a new species, for which the name S. tritolerans is proposed, with strain DAS 165(T) as the type strain (=DSM 41899(T )= CCTCCAA 206013(T)).     

<Emphasis Type="Italic">Streptomyces gulbargensis</Emphasis> sp. nov., isolated from soil in Karnataka,India

During the course of screening for industrially important microorganisms, an alkali-tolerant and thermotolerant actinomycete, strain DAS 131T, was isolated from a soil sample collected from the Gulbarga region, Karnataka province, India. The strain was characterized by a polyphasic approach that showed that it belonged to the genus Streptomyces. Growth was observed over a wide pH range (pH 6-12) and at 45 degrees C. The 16S rRNA gene sequence of strain DAS 131T was deposited in the GenBank database under the accession number DQ317411. 16S rRNA gene sequence analysis revealed that strain DAS 131T was most closely related to Streptomyces venezuelae ISP 5230T (AY999739) with a sequence similarity of 99.5% (8 nucleotide differences out of 1,477). Despite this very high sequence similarity, strain DAS 131T was phenetically distinct from S. venezuelae. The DNA relatedness between these strains was 54%, indicating that strain DAS 131T is a distinct genomic species. On the basis of phenetic and genetic analyses, strain DAS 131T is classified as a new species in the genus Streptomyces, for which we propose the name Streptomyces gulbargensis sp. nov.     

Adding some SPICE to DAS

The distributed annotation system (DAS) defines a communication protocol used to exchange biological annotations. It is motivated by the idea that annotations should not be provided by single centralized databases but instead be spread over multiple sites. Data distribution, performed by DAS servers, is separated from visualization, which is carried out by DAS clients. The original DAS protocol was designed to serve annotation of genomic sequences. We have extended the protocol to be applicable to macromolecular structures. Here we present SPICE, a new DAS client that can be used to visualize protein sequence and structure annotations. AVAILABILITY: http://www.efamily.org.uk/software/dasclients/spice/     

Characterization and sequence prediction of structural variations in α-helix

Background

Centralised resources such as GenBank and UniProt are perfect examples of the major international efforts that have been made to integrate and share biological information. However, additional data that adds value to these resources needs a simple and rapid route to public access. The Distributed Annotation System (DAS) provides an adequate environment to integrate genomic and proteomic information from multiple sources, making this information accessible to the community. DAS offers a way to distribute and access information but it does not provide domain experts with the mechanisms to participate in the curation process of the available biological entities and their annotations.

Results

We designed and developed a Collaborative Annotation System for proteins called DAS Writeback. DAS writeback is a protocol extension of DAS to provide the functionalities of adding, editing and deleting annotations. We implemented this new specification as extensions of both a DAS server and a DAS client. The architecture was designed with the involvement of the DAS community and it was improved after performing usability experiments emulating a real annotation task.

Conclusions

We demonstrate that DAS Writeback is effective, usable and will provide the appropriate environment for the creation and evolution of community protein annotation.     

Chemical genetic identification of glutamine phosphoribosylpyrophosphate amidotransferase as the target for a novel bleaching herbicide in Arabidopsis

A novel phenyltriazole acetic acid compound (DAS734) produced bleaching of new growth on a variety of dicotyledonous weeds and was a potent inhibitor of Arabidopsis (Arabidopsis thaliana) seedling growth. The phytotoxic effects of DAS734 on Arabidopsis were completely alleviated by addition of adenine to the growth media. A screen of ethylmethanesulfonate-mutagenized Arabidopsis seedlings recovered seven lines with resistance levels to DAS734 ranging from 5- to 125-fold. Genetic tests determined that all the resistance mutations were dominant and allelic. One mutation was mapped to an interval on chromosome 4 containing At4g34740, which encodes an isoform of glutamine phosphoribosylamidotransferase (AtGPRAT2), the first enzyme of the purine biosynthetic pathway. Sequencing of At4g34740 from the resistant lines showed that all seven contained mutations producing changes in the encoded polypeptide sequence. Two lines with the highest level of resistance (125-fold) contained the mutation R264K. The wild-type and mutant AtGPRAT2 enzymes were cloned and functionally overexpressed in Escherichia coli. Assays of the recombinant enzyme showed that DAS734 was a potent, slow-binding inhibitor of the wild-type enzyme (I(50) approximately 0.2 microm), whereas the mutant enzyme R264K was not significantly inhibited by 200 microm DAS734. Another GPRAT isoform in Arabidopsis, AtGPRAT3, was also inhibited by DAS734. This combination of chemical, genetic, and biochemical evidence indicates that the phytotoxicity of DAS734 arises from direct inhibition of GPRAT and establishes its utility as a new and specific chemical genetic probe of plant purine biosynthesis. The effects of this novel GPRAT inhibitor are compared to the phenotypes of known AtGPRAT genetic mutants.     

A cross-sectional study of pain sensitivity,disease-activity assessment,mental health,and fibromyalgia status in rheumatoid arthritis

IntroductionPain remains the most important problem for people with rheumatoid arthritis (RA). Active inflammatory disease contributes to pain, but pain due to non-inflammatory mechanisms can confound the assessment of disease activity. We hypothesize that augmented pain processing, fibromyalgic features, poorer mental health, and patient-reported 28-joint disease activity score (DAS28) components are associated in RA.MethodsIn total, 50 people with stable, long-standing RA recruited from a rheumatology outpatient clinic were assessed for pain-pressure thresholds (PPTs) at three separate sites (knee, tibia, and sternum), DAS28, fibromyalgia, and mental health status. Multivariable analysis was performed to assess the association between PPT and DAS28 components, DAS28-P (the proportion of DAS28 derived from the patient-reported components of visual analogue score and tender joint count), or fibromyalgia status.ResultsMore-sensitive PPTs at sites over or distant from joints were each associated with greater reported pain, higher patient-reported DAS28 components, and poorer mental health. A high proportion of participants (48%) satisfied classification criteria for fibromyalgia, and fibromyalgia classification or characteristics were each associated with more sensitive PPTs, higher patient-reported DAS28 components, and poorer mental health.ConclusionsWidespread sensitivity to pressure-induced pain, a high prevalence of fibromyalgic features, higher patient-reported DAS28 components, and poorer mental health are all linked in established RA. The increased sensitivity at nonjoint sites (sternum and anterior tibia), as well as over joints, indicates that central mechanisms may contribute to pain sensitivity in RA. The contribution of patient-reported components to high DAS28 should inform decisions on disease-modifying or pain-management approaches in the treatment of RA when inflammation may be well controlled.     

Decrease of hepatic catalase level by treatment with diallyl sulfide and garlic homogenates in rats and mice

Diallyl sulfide (DAS) is a flavor compound derived from garlic and is active in the inhibition of chemically induced cytotoxicity and carcinogenicity in animal models. This study was conducted to examine the effects of the treatment of DAS and garlic homogenates on the activities of catalase, glutathione peroxidase, and superoxide dismutase. Male Sprague-Dawley rats were treated with DAS i.g. at daily doses of 50 or 200 mg/kg for 8 days, causing the hepatic catalase activity to decrease by 55 and 95%, respectively. Such a decrease in hepatic catalase activity was also observed when the DAS treatment was extended to 29 days. Western blot analysis showed that the DAS treatments resulted in corresponding decreases in the liver catalase protein level. No significant change in the catalase activity in the kidney, lung, and brain was observed with the treatments, but a slight decrease in heart catalase activity was observed. These treatments did not cause significant changes in superoxide dismutase and glutathione peroxidase activities in these tissues. Treatment with DAS at a daily dose of 200 mg/kg for 1-7 days resulted in a gradual decrease in the liver catalase activity to 5% of the control level, but it did not decrease the erythrocyte catalase activity. Treatment of rats with fresh garlic homogenates (2 or 4 g/kg, i.g., daily for 7 days) caused a 35% decrease in liver catalase activity. A/J mice treated with DAS and garlic homogenates also showed a decrease in the liver catalase activity. Diallyl sulfone (DASO2), a DAS metabolite, however, did not effectively decrease catalase activity in mice. The catalase activity was not inhibited by either DAS or DASO2 in vitro. The present results demonstrate that treatment with DAS and garlic homogenates decrease the hepatic catalase level in rats and mice.     

Metabolic responses to change in disease activity during tumor necrosis factor inhibition in patients with rheumatoid arthritis

Assessment of disease activity in patients with rheumatoid arthritis (RA) is of importance in the evaluation of treatment. The most important measure of disease activity is the Disease Activity Score counted in 28 joints (DAS28). In this study, we evaluated whether metabolic profiling could complement current measures of disease activity. Fifty-six patients, in two separate studies, were followed for two years after commencing anti-TNF therapy. DAS28 was assessed, and metabolic profiles were recorded at defined time points. Correlations between metabolic profile and DAS28 scores were analyzed using multivariate statistics. The metabolic responses to lowering DAS28 scores varied in different patients but could predict DAS28 scores at the individual and subgroup level models. The erythrocyte sedimentation rate (ESR) component in DAS28 was most correlated to the metabolite data, pointing to inflammation as the primary effect driving metabolic profile changes. Patients with RA had differing metabolic response to changes in DAS28 following anti-TNF therapy. This suggests that discovery of new metabolic biomarkers for disease activity will derive from studies at the individual and subgroup level. Increased inflammation, measured as ESR, was the main common effect seen in metabolic profiles from periods associated with high DAS28.     

Delayed anovulatory syndrome induced by estradiol in female C57BL/6J mice: age-like neuroendocrine, but not ovarian, impairments

These studies describe induction of a delayed anovulatory syndrome (DAS) by estradiol (E2) in female C57BL/6J mice. Six days after birth, female mice were injected s.c. with 0.1 micrograms estradiol benzoate or oil. Over 90% of the oil-injected controls exhibited estrous cycles from 2 to 9 mo of age. In contrast, 60% of the E2-injected mice exhibited estrous cycles at 2 mo of age but were acyclic by 9 mo; these mice were considered to have exhibited a DAS, and had longer cycles than controls. At 12 mo, ovarian impairments were assessed by examining 1) ovulation after s.c. injection of 5 IU human chorionic gonadotropin (hCG), and 2) estrous cycles after grafting into young (3-mo-old) hosts. Simultaneously, neuroendocrine impairments were assessed by examining 1) the surge of luteinizing hormone (LH) induced by E2 implants after ovariectomy, and 2) estrous cycles after receiving ovarian grafts from 3-mo-old mice. Ovaries from DAS and control mice ovulated equally in response to hCG. Ovaries from DAS mice grafted into young ovariectomized hosts supported 30% more cycles, of shorter period, compared with ovaries from control donors. However, the E2-induced LH surge was 50% smaller in DAS mice than in controls. Ovariectomized DAS hosts with ovarian grafts from young mice supported 70% fewer estrous cycles, of longer period, compared with ovariectomized control hosts with young grafts. We conclude that the E2-induced DAS in female mice is not due to ovarian impairments, but seems to result from neuroendocrine impairments.     

Influence of metabolic and physical factors on production of diacetoxyscirpenol by Fusarium sambucinum Fuckel.

Fusarium sambucinum Fuckel 8099-1 was grown on Czapek-Dox peptone-supplemented medium at 15 degrees C for 14 days, and the cultures were investigated for diacetoxyscirpenol (DAS) production by liquid-liquid extraction and gas chromatography. The addition of 150 mg of sorbic acid, a tricarboxylic acid cycle inhibitor, per liter stimulated both fungal growth and DAS production. Among the beta-hydroxy-beta-methylglutaryl coenzyme A precursors tested, isovaleric acid completely inhibited fungal growth and DAS production, ethyl isovalerate did not support a significant increase in DAS production, and L-leucine partially inhibited DAS production, showing that L-leucine and isovaleric acid catabolisms do not induce trichothecene biosynthesis. Solid particles (cork powder) were necessary for DAS production in stationary cultures but did not influence DAS production in shaken cultures. Shaking strongly stimulated DAS production and fungal growth.     

Influence of metabolic and physical factors on production of diacetoxyscirpenol by Fusarium sambucinum Fuckel

Fusarium sambucinum Fuckel 8099-1 was grown on Czapek-Dox peptone-supplemented medium at 15 degrees C for 14 days, and the cultures were investigated for diacetoxyscirpenol (DAS) production by liquid-liquid extraction and gas chromatography. The addition of 150 mg of sorbic acid, a tricarboxylic acid cycle inhibitor, per liter stimulated both fungal growth and DAS production. Among the beta-hydroxy-beta-methylglutaryl coenzyme A precursors tested, isovaleric acid completely inhibited fungal growth and DAS production, ethyl isovalerate did not support a significant increase in DAS production, and L-leucine partially inhibited DAS production, showing that L-leucine and isovaleric acid catabolisms do not induce trichothecene biosynthesis. Solid particles (cork powder) were necessary for DAS production in stationary cultures but did not influence DAS production in shaken cultures. Shaking strongly stimulated DAS production and fungal growth.     

Appearance of different phosphatase forms and phosphorus partitioning in nodules of chickpea (Cicer arietinum L.) during development

Acid and alkaline phosphatase and phytase activities were determined in the bacteroid free fractions of chickpea (Cicer arietinum L.) nodules at 15 days intervals, from 40 days after sowing (DAS) to 85 DAS. In general, the activities and specific activity of both the acid and alkaline phosphatases declined at 55 DAS. Out of the various substrates studied, ATP was the best substrate for both phosphatases. Activities of phosphatases with glucose-6-phosphate and fructose-6-phosphate were low in comparison to these with fructose 1,6 bisphosphate. The efficiency of acid phosphatase for utilizing fructose 1,6 bis phosphate as a substrate increased with nodule development. A fructose 1,6 bis phosphate specific acid phosphatase with elution volume to void volume (Ve/Vo) ratio of around 2.0 was observed in mature nodules (80 DAS). Acid phosphatase at 40 DAS was resolved into two peaks which were eluted at Ve/Vo of about 1.5 and 1.8. However, at 60 DAS the peak with Ve/Vo of 1.5 could not be detected. With ATP as substrate, a high (Ve/Vo of 1.2) and low MM form (Ve/Vo of 2.1) alkaline phosphatases were observed at 40 DAS however at 60 DAS stage only one peak with Ve/Vo of 1.7 was detected. Although, a low activity of acid phytase was observed in nodules at all stages of development but neither alkaline phytase nor phytic acid could be detected. It appears that the nodules acquire inorganic phosphate from the roots. The higher content of water soluble organic phosphorus in mature nodules could be due to the low activities of phosphatases at maturity.     

Estimating western corn rootworm (Coleoptera: Chrysomelidae) larval susceptibility to event DAS‐59122‐7 maize

Susceptibility of Diabrotica virgifera virgifera (LeConte) larvae to DAS‐59122‐7 maize was evaluated using a laboratory technique that measures rootworm survival to adulthood on maize seedlings. This method produces direct measures of larval susceptibility using realistic exposure to the same range of insecticidal protein concentrations found in field‐grown DAS‐59122‐7 maize roots. First, second and third instars were reared to adulthood on DAS‐59122‐7 maize seedlings or a non‐transgenic, near‐isoline maize. Data on survival, adult gender ratio, adult weight and median emergence were collected. Overall, larval susceptibility to DAS‐59122‐7 maize was lower than earlier predictions ( Storer et al. 2006 ). Neonate survival on DAS‐59122‐7 maize was approximately 33% of isoline survival after 17 days, and the same 33% recovered and developed to adulthood when the isoline maize was substituted. Survival rate on DAS‐59122‐7 maize increased with instar. The mean survivorship was 0.5%, 26% and 65% when exposure to DAS‐59122‐7 maize began at the first, second and third instars, respectively. Exposure to DAS‐59122‐7 maize led to sub‐lethal effects on adult gender ratio, weight and median emergence. These effects decreased when exposure to DAS‐59122‐7 maize began at later instars. The killing effect of DAS‐59122‐7 maize on rootworm larvae appeared to result from the combined chronic effects and absence of a suitable host as perceived by the larvae. The relevance of these data and the methodology of estimating rootworm susceptibility to plant‐incorporated protectants are discussed in the context of the US Environmental Protection Agency’s functional definition of ‘high dose’ and use of refuge for resistance management ( EPA 1998a ). Based on these results it is evident that DAS‐59122‐7 maize does not meet the functional definitions of high dose as described by EPA (1998a,b) and ILSI (1999) , and the utility of refuge, refuge size and refuge placement for delaying rootworm resistance should be further investigated.     

Effect of hydro- and osmopriming of chickpea (Cicer arietinum L.) seeds on enzymes of sucrose and nitrogen metabolism in nodules

Three year data on the effect of water- and mannitol (4%) priming of chickpea seeds (12 h at 25°C) showed higher number and biomass of nodules in the plants from primed seeds than from non-primed seeds. The biomass of nodules increased to 75 DAS but decreased by 90 DAS. Activities of sucrose metabolism enzymes (sucrose synthase (SS) and alkaline invertase) and of nitrogen metabolism (glutamine synthetase (GS), glutamate synthase (GOGAT) and glutamate dehydrogenase (GDH)) in nodules of primed and non-primed crops during development are reported. SS and alkaline invertase activities increased to 70 DAS and then decreased. In primed plants, the higher SS activity in nodules at 60 and 70 DAS might be responsible for providing more energy and carbon skeleton for nitrogen fixation and for ammonium assimilation in primed plants. At 85 DAS, though the SS activity decreased in comparison with the earlier growth stages, it was still higher in nodules of the primed crops than the non-primed crop. Activity of alkaline invertase was maximum at 70 DAS in the nodules of primed and non-primed crops. Priming increased nodule GS activity at 70 and 85 DAS. GOGAT activity was unaffected by priming but GDH activity was greater in nodules from primed crops at 50 DAS. Elevated SS and GS nodule activities in primed chickpeas might be responsible in increasing nodule biomass and metabolic activity thereby increasing seed fill.     

Diallyl sulfide induces apoptosis in Colo 320 DM human colon cancer cells: involvement of caspase-3, NF-κB, and ERK-2

Chemoprevention is regarded as one of the most promising and realistic approaches in the prevention of human cancer. Diallyl sulfide (DAS), an organosulfur component of garlic has been known for its chemopreventive activities against various cancers and also in recent years, numerous investigations have shown that sulfur-containing compounds induce apoptosis in multiple cell lines and experimental animals. Thus the present study was focused to elucidate the anticancerous effect and the mode of action of DAS against Colo 320 DM colon cancer cells. DAS induced apoptosis in Colo 320 DM cells was revealed by flow cytometer analysis and phosphatidyl serine exposure. DAS also promoted cell cycle arrest substantially at G2/M phase in Colo 320 DM cells. The production of reactive oxygen intermediates, which were examined by 2,7-dichlorodihydrofluorescein diacetate (H₂DCF-DA), increased with time, after treatment with DAS. The activities of alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) were decreased upon DAS treatment, which shows the antiproliferative and the cytotoxic effects, respectively. The expression of NF-κB was upregulated in DAS treated cells, compared to normal cells. Further, DAS promoted the expression of caspase-3 and suppression of Extracellular Regulatory Kinase-2 (ERK-2) activity in Colo 320 DM cells that was determined by Western blot analysis. In conclusion, DAS increased the production of ROS, caused cell cycle arrest, decreased cell proliferation and induced apoptosis in Colo 320 DM cells. Thus, this study put forward DAS as a drug that can possibly be used to treat cancers.     

A new semisynthetic derivative of sauroine induces LTP in hippocampal slices and improves learning performance in the Morris Water Maze

Two semisynthetic acetyl derivatives of the alkaloid sauroine from Huperzia saururus, monoacetyl sauroine, and diacetyl sauroine (DAS) were obtained and their chemical structures were analyzed by NMR. While monoacetyl sauroine is the typical product of acetylation, DAS is an unexpected derivative related to the keto‐enol formation of sauroine. Recordings of field excitatory post‐synaptic potentials from the CA1 region of rat hippocampal slices showed that only DAS acutely applied induced chemical long‐term potentiation (LTP) in a dose‐dependent manner with an EC₅₀ of 1.15 ± 0.09 μM. This effect was blocked by 10 μM D(‐)‐2‐amino‐5‐phosphonopentanoic acid (AP5), suggesting dependence on the NMDA receptor. DAS significantly increased NMDA receptor‐dependent excitatory post‐synaptic currents without affecting α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate receptor‐dependent currents. Repetitive administration of DAS improved visuo‐spatial learning in the Morris Water Maze. In slices from rats tested in the Morris Water Maze, LTP resulting from electrical synaptic stimulation was 2.5 times larger than in controls. Concentration of DAS measured in the brain after repetitive administration was 29.5 μM. We conclude that slices perfused with DAS display a robust NMDA receptor‐dependent chemical LTP. During chronic treatment, DAS enhances learning abilities through a metaplastic mechanism as revealed by the augmentation of LTP in slices. DAS, therefore, may be a promising compound as a nootropic therapeutic drug.

     

Effect of High Temperature Stress at Anthesis on Grain Yield and Biomass of Field-grown Crops of Wheat

Spring wheat (Triticum aestivumL., ‘Chablis’) wasgrown under field conditions from sowing until harvest maturity,except for a 12-d period [70–82 days after sowing (DAS)coinciding with anthesis] during which replicated crop areaswere exposed to a range of temperatures within two pairs ofpolyethylene-covered temperature gradient tunnels. At 82 DAS,an increase in mean temperature from 16 to 25 °C duringthis treatment period had no effect on above-ground biomass,but increased ear dry weight from 223 to 327 g m^-2and, at 83DAS, reduced root biomass from 141 to 63 g m^-2. Mean temperatureover the treatment period had no effect on either above-groundbiomass or grain yield at maturity. However, the number of grainsper ear at maturity declined with increasing maximum temperaturerecorded over the mid-anthesis period (76–79 DAS) and,more significantly, with maximum temperature 1 d after 50% anthesis(78 DAS). Grain yield and harvest index also declined sharplywith maximum temperature at 78 DAS. Grain yield declined by350 g m^-2at harvest maturity with a 10 °C increase in maximumtemperature at 78 DAS and was related to a 40% reduction inthe number of grains per ear. Grain yield was also negativelyrelated to thermal time accumulated above a base temperatureof 31 °C (over 8 d of the treatment from 5 d before to 2d after 50% anthesis). Thus, grain fertilization and grain setwas most sensitive to the maximum temperature at mid-anthesis.These results confirm that wheat yields would be reduced considerablyif, as modellers suggest, high temperature extremes become morefrequent as a result of increased variability in temperatureassociated with climate change.Copyright 1998 Annals of BotanyCompany Triticum aestivum, spring wheat, temperature, grain number, grain yield, root growth.     

Reversible inhibitions of gastric H+,K(+)-ATPase by scopadulcic acid B and diacetyl scopadol. New biochemical tools of H+,K(+)-ATPase

Scopadulcic acid B (SA-B), a novel diterpenoid, is a main ingredient of the Paraguayan traditional medicinal herb "Typychá kuratú (Scoparia dulcis L.). SA-B and its debenzoyl derivative, diacetyl scopadol (DAS), specifically inhibit ATP hydrolysis of gastric H+,K(+)-ATPase. Both compounds inhibit the K(+)-dependent dephosphorylation step of the enzyme without any effect on the phosphorylation step. SA-B is a mixed-type inhibitor with respect to the activating cation, K+. SA-B lowers the affinity of H+,K(+)-ATPase to K+ and decreases the maximal velocity of ATP hydrolysis, whereas DAS is an uncompetitive inhibitor with respect to K+. Furthermore, the effects of SA-B and DAS on conformational states of the ATPase were studied by measuring the changes in the fluorescence intensity of the fluorescein isothiocyanate-labeled enzyme. The fluorescence study shows that SA-B primarily binds to the E2K form in the presence of Mg2+ and stabilizes the form and that DAS stabilizes the E2PK form. Therefore, the chemical modification of SA-B, debenzoylation, induced the changes in the pattern of inhibition of H+,K(+)-ATPase. Furthermore, the inhibition mechanisms of SA-B and DAS were different from those of omeprazole, which is an irreversible inhibitor, and SCH 28080, which is a reversible, competitive inhibitor with respect to K+. DAS also inhibited the K(+)-dependent p-nitrophenyl phosphatase activity, and the inhibition was competitive with respect to K+, indicating that the K(+)-dependent p-nitrophenylphosphatase activity does not represent the partial reaction step of H+,K(+)-ATPase.